Structural studies of the polysaccharide antigen of Eubacterium saburreum, Strain L 32

The structure of the polysaccharide antigen produced by Eubacterium saburreum, strain L 32, has been investigated. The principal methods used were methylation analysis, graded hydrolysis with acid, and n.m.r. spectroscopy. The polysaccharide, which contains the unusual sugar 3,6-dideoxy-D-arabino-hexose (tyvelose, Tyv), is composed of trisaccharide repeating-units having the following structure:

     

Structural studies of the polysaccharide antigen of Eubacterium saburreum, strain L. 452

The structure of the polysaccharide antigen produced by Eubacterium saburreum, strain L 452, has been investigated. Methylation analysis, graded hydrolysis with acid, and n.m.r. spectroscopy were the principal methods used. The polysaccharide is composed of trisaccharide repeating-units having the following structure:

The assignment of the β configuration to the d-ribofuranosyl residue is tentative.     

Photochemical cycloaddition of 1,3-diacetoxy-2-propanone to (trimethylsilyloxy)ethylene

The structures of two capsular polysaccharides elaborated by Haemophilus influenzae type e, strains NCTC 8455 and 8472, respectively, have been investigated, methylation analysis and n.m.r. spectrometry being the principal methods used. It is concluded that the polysaccharides are composed of repeating-units having the following structure:

In the polysaccharide from strain NCTC 8472, all of the repeating-units contain the β-dfructofuranosyl group. The polysaccharide from strain NCTC 8455, however, contains only traces of d-fructose, corresponding to approximately one group per 100 repeating-units.     

Structural studies of the O-antigen polysaccharide of Escherichia coli O4

The structure of the O-antigen polysaccharide of Escherichia coli O4 has been investigated using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the O-antigen is composed of pentasaccharide repeating-units having the following structure.

This structure differs in some details from that recently proposed by Schmidt et al.     

Structure of the extracellular polysaccharide from xanthomonas campestris

Xanthan gum, the extracellular polysaccharide from Xanthomonas campestris, has been reinvestigated by methylation analysis, and by uronic acid degradation followed by oxidation and elimination of the oxidized residue. The polysaccharide is composed of pentasaccharide repeating-units with the following structure:

     

Structural studies of the capsular polysaccharide of Klebsiella type 37

The structure of the Klebsiella type 37 capsular polysaccharide has been investigated. Methylation analysis, various specific degradations, and n.m.r. spectroscopy were the principal methods used. It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the structure 4-O-Lac-d-GlcA  4-O-[(S)-1-carboxyethyl]-d-glucuronic acid:

     

Structural studies of gellan gum,an extracellular polysaccharide elaborated by Pseudomonas elodea

The structure of gellan gum, a polysaccharide of potential commercial usefulness elaborated by Pseudomonas elodea, has been investigated. It is concluded that the polysaccharide is composed of tetrasaccharide repeating-units having the following structure.

Of the repeating units, ～25% contain an O-acetyl group linked to C-6 of one of the β-d-glucopyranosyl residues.     

Structural studies of the capsular antigen from streptococcus pneumoniae type 26

The structure of the capsular antigen from Pneumococcus type 26 has been determined by using methylation analysis, periodate-oxidation studies, and n.m.r. spectroscopy of the original and the dephosphorylated product. It is concluded that the polysaccharide is composed of repeating-units having the following structure.

The only difference between this structure and that of the type-6 antigen is that the α-l-rhamnopyranosyl residue is linked to O-4 of d-ribitol in the former, but to O-3 in the latter.     

Structural studies of the O-specific side-chain of the lipopolysaccharide from Escherichia coli O 55

The structure of the O-specific side-chains of the lipopolysaccharide from Escherichia coli O 55 has been investigated, methylation analysis, specific degradations, and n.m.r. spectroscopy being the principal methods used. It is concluded that the O-specific side-chains are composed of pentasaccharide repeating-units having the following structure [where Col stands for colitose (3,6-dideoxy-l-xylo-hexose)].

     

Structural studies of the Rhizobium trifolii extracellular polysaccharide

The structure of the extracellular polysaccharide of Rhizobium trifolii has been investigated. Methylation analysis, sequential degradations by oxidation and elimination of oxidized residues, uronic acid degradation, and degradation by oxidation of the acetylated polysaccharide with chromium trioxide in acetic acid were the main methods used. It is proposed that the polysaccharide is composed of heptasaccharide repeating-units having the following structure:

An unusual feature is that some of the repeating units are incomplete and lack the terminal β-d-galactopyranosyl group. The polysaccharide contains O-acetyl groups (somewhat more than 1 mol. per unit), linked to O-2 and O-3 of 4-O-substituted d-glucopyranosyl chain-residues. A previous finding that the polysaccharide contains 2-deoxy-d-arabino-hexose (2-deoxy-d-glucose) residues is erroneous.     

A Novel Glucosyltransferase Involved in O-Antigen Modification of Shigella flexneri Serotype 1c

The O antigen of serotype 1c differs from the unmodified O antigen of serotype Y by the addition of a disaccharide (two glucosyl groups) to the tetrasaccharide repeating unit. It was shown here that addition of the first glucosyl group is mediated by the previously characterized gtrI cluster, which is found within a cryptic prophage at the proA locus in the bacterial chromosome. Transposon mutagenesis was performed to disrupt the gene responsible for addition of the second glucosyl group, causing reversion to serotype 1a. Colony immunoblotting was used to identify the desired revertants, and subsequent sequencing, cloning, and functional expression successfully identified the gene encoding serotype 1c-specific O-antigen modification. This gene (designated gtrIC) was present as part of a three-gene cluster, similar to other S. flexneri glucosyltransferase genes. Relative to the other S. flexneri gtr clusters, the gtrIC cluster is more distantly related and appears to have arrived in S. flexneri from outside the species. Analysis of surrounding sequence suggests that the gtrIC cluster arrived via a novel bacteriophage that was subsequently rendered nonfunctional by a series of insertion events.Shigella flexneri is a pathovar of Escherichia coli that is the main causative agent of endemic bacillary dysentery (shigellosis). It is estimated that S. flexneri is responsible for approximately 100 million shigellosis cases annually, resulting in hundreds of thousands of deaths, predominantly in young children (11). Currently no vaccine is available, although there is evidence to suggest that serotype-specific immunity occurs following infection and that induction of immunity can be replicated with vaccines (9). Shigella serotype diversity arises due to differences in the chemical structure of the O-antigen repeating unit in the lipopolysaccharide, which is the main target of the adaptive host immune response following infection.Because immunity to S. flexneri can be conferred by the induction of antibodies directed against the O antigen, an understanding of the prevalence of different serotypes and the underlying basis of serotype diversity can inform appropriate vaccine design. All S. flexneri serotypes (with the exception of serotype 6) share a common O-antigen backbone, consisting of a repeating tetrasaccharide unit that is comprised of one N-acetylglucosamine residue (GlcNAc) and three rhamnose residues (RhaI, RhaII, and RhaIII) (14). The 12 traditionally recognized S. flexneri serotypes differ by the presence or absence of just six different chemical modifications (glucosylations or O acetylations) of the O antigen. The genes responsible for these O-antigen modifications are introduced into the bacterial genome via bacteriophages (3). Glucosylation of the S. flexneri O antigen is mediated by three genes [gtrA, gtrB, and gtr(type)] that are arranged in a single operon known as a gtr cluster. gtrA and gtrB are highly conserved between different gtr clusters and encode proteins involved in transferring the glucosyl group from the cytoplasm into the periplasm, where O-antigen modification is thought to take place. gtr(type) is unique to each gtr cluster and encodes a glucosyltransferase that is responsible for attaching the glucosyl group to a specific sugar unit of the O antigen via a specific linkage (3).Investigations of S. flexneri have typically focused on serotypes for which commercially available typing sera are available. More recently, it has become clear that other serotypes are also epidemiologically important. In Bangladesh in the late 1980s, two novel S. flexneri strains that did not agglutinate with antibodies specific for the traditionally recognized serotypes were isolated (4). Chemical analysis of the O antigen revealed that these strains belonged to a new serotype, which was named serotype 1c due to the similarity its O antigen shares with the O antigens of serotype 1a and 1b strains (19). Serotype 1c has since been isolated in Egypt, Indonesia, Pakistan, and Vietnam (6, 15, 18). Serotype 1c was shown to be the most prevalent S. flexneri serotype in a northern province of Vietnam, accounting for more than a third of all S. flexneri strains isolated from 1998 to 1999 (15). Identification of serotype 1c currently relies on agglutination testing using monoclonal antibody MASF Ic (19).The O antigen of serotype 1c is distinguished by the presence of a disaccharide (two glucosyl groups) linked to the GlcNAc in the tetrasaccharide repeating unit of the O antigen. The first glucosyl group is joined to GlcNAc via an α1→4 linkage, as occurs in the O antigen of serotype 1a and serotype 1b strains (type I modification). The O antigen of serotype 1c is distinguished by the presence of a second glucosyl group that is linked to the first via an α1→2 linkage (Fig. ​(Fig.1).1). Type Ia modification is prerequisite to type Ic modification.Open in a separate windowFIG. 1.Chemical structure of the tetrasaccharide repeat units in the O antigens of S. flexneri serotypes 1a and 1c. Note that the O antigen of serotype 1b (not shown) differs from that of serotype 1a by the O acetylation of l-RhaIII.In this study, the genetic basis of O-antigen modification in serotype 1c was elucidated. Serotype 1c strains isolated from different locations and times were compared to gain insight into the evolution of this serotype. This is the first report of the identification of a glucosyltransferase gene that is responsible for addition of the second glucosyl group, causing serotype conversion from serotype 1a to serotype 1c.     

Structure of the O-antigen of Yersinia pseudotuberculosis O:4a revised

The O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Yersinia pseudotuberculosis O:4a and studied by NMR spectroscopy, including 2D ROESY and ¹H, ¹³C HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established, which differs from the structure reported earlier [Gorshkova, R. P. et al., Bioorg. Khim. 1983, 9, 1401-1407] in the linkage modes between the monosaccharides: where Tyv stands for 3,6-dideoxy-d-arabino-hexose (tyvelose). The structure of the Y. pseudotuberculosis O:4a antigen resembles that of Y. pseudotuberculosis O:2c, which differs in the presence of abequose (3,6-dideoxy-d-xylo-hexose) in place of tyvelose only.     

Variable lymphocyte receptor recognition of the immunodominant glycoprotein of Bacillus anthracis spores

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Highlights? VLRBs use their C-terminal LRRs and the LRRCT-loop to interact with antigen ? Sequence-related VLRBs exhibit differential recognition of their BclA epitopes ? VLR4 binds a conserved protein epitope, yet is specific for B. anthracis spores     

Genetic Analysis for the Lack of Expression of the O157 Antigen in an O Rough:H7 Escherichia coli Strain

The O-antigen (rfb) operon and related genes of MA6, an O rough:H7 Shiga-toxigenic Escherichia coli strain, were examined to determine the cause of the lack of O157 expression. A 1,310-bp insertion, homologous to IS629, was observed within its gne gene. trans complementation with a functional gne gene from O157:H7 restored O157 antigen expression in MA6.Shiga-toxigenic Escherichia coli (STEC) serotype O157:H7 carries O157 and H7 antigens, so these traits are extensively used in identification (1). Strain MA6, isolated from beef in Malaysia (8), carries the O157:H7 virulence factor genes, including the Shiga toxin 2 gene (stx₂), the γ intimin allele (γ-eae), the enterohemolysin gene (ehxA), and the +93 uidA single nucleotide polymorphism (SNP) found only in O157:H7 strains (1). Multilocus sequence typing also showed MA6 to have the most common sequence type (ST-66) for O157:H7 strains. However, and in spite the fact that MA6 had per gene sequences essential for O157 antigen synthesis (2), no O157 antigen is expressed (O rough), and therefore, it is undetectable with serological assays used in O157:H7 analysis.The biosynthesis and assembly of E. coli O antigen are highly complex (9). The rfb operon (12 genes) (16), along with 3 ancillary genes outside of the rfb, is required for the biosynthesis of the 4 sugar nucleotide precursors and the assembly of the O unit (11). This is then linked to the core antigen, comprising an inner and an outer component, which require 3 other operons for biosynthesis and assembly (9). As defects in any of these genes could produce the O-null phenotype (13), we systematically examined these genes (Table ​(Table1)1) to elucidate the cause of the absence of O157 expression in MA6.

TABLE 1.

rfb operon genes, ancillary genes, and waa cluster genes examined in this study

Structures of the O-antigens of Escherichia coli O13, O129, and O135 related to the O-antigens of Shigella flexneri

O-Polysaccharides (O-antigens) were isolated from Escherichia coli O13, O129, and O135 and studied by chemical analyses along with 2D ¹H and ¹³C NMR spectroscopy. They were found to possess a common →2)-l-Rha-(α1→2)-l-Rha-(α1→3)-l-Rha-(α1→3)-d-GlcNAc-(β1→ backbone, which is a characteristic structural motif of the O-polysaccharides of Shigella flexneri types 1-5. In both the bacterial species, the backbone is decorated with lateral glucose residues or/and O-acetyl groups. In E. coli O13, a new site of glycosylation on 3-substituted Rha was revealed and the following O-polysaccharide structure was established:The structure of the E. coli O129 antigen was found to be identical to the O-antigen structure of S. flexneri type 5a specified in this work and that of E. coli O135 to S. flexneri type 4b reported earlier.     

Structural investigation of the capsular polysaccharide of Escherichia coli O101 : K103 : H− using bacteriophage degradation and NMR spectroscopy

NMR spectroscopy was performed on the depyruvated capsular antigen of E. coli K103 and on the oligosaccharide obtained by depolymerisation of the native polysaccharide with a viral-borne endoglycanase. This capsular polysaccharide is the only one to be co-expressed with O group 101 and joins a small group of unusual capsular polysaccharides which possess pyruvic acid as the only acidic function. The primary structure was shown to be composed of the repeating unit:

     

Phase Ia clinical evaluation of the safety and immunogenicity of the Plasmodium falciparum blood-stage antigen AMA1 in ChAd63 and MVA vaccine vectors

Background

Traditionally, vaccine development against the blood-stage of Plasmodium falciparum infection has focused on recombinant protein-adjuvant formulations in order to induce high-titer growth-inhibitory antibody responses. However, to date no such vaccine encoding a blood-stage antigen(s) alone has induced significant protective efficacy against erythrocytic-stage infection in a pre-specified primary endpoint of a Phase IIa/b clinical trial designed to assess vaccine efficacy. Cell-mediated responses, acting in conjunction with functional antibodies, may be necessary for immunity against blood-stage P. falciparum. The development of a vaccine that could induce both cell-mediated and humoral immune responses would enable important proof-of-concept efficacy studies to be undertaken to address this question.

Methodology

We conducted a Phase Ia, non-randomized clinical trial in 16 healthy, malaria-naïve adults of the chimpanzee adenovirus 63 (ChAd63) and modified vaccinia virus Ankara (MVA) replication-deficient viral vectored vaccines encoding two alleles (3D7 and FVO) of the P. falciparum blood-stage malaria antigen; apical membrane antigen 1 (AMA1). ChAd63-MVA AMA1 administered in a heterologous prime-boost regime was shown to be safe and immunogenic, inducing high-level T cell responses to both alleles 3D7 (median 2036 SFU/million PBMC) and FVO (median 1539 SFU/million PBMC), with a mixed CD4⁺/CD8⁺ phenotype, as well as substantial AMA1-specific serum IgG responses (medians of 49 µg/mL and 41 µg/mL for 3D7 and FVO AMA1 respectively) that demonstrated growth inhibitory activity in vitro.

Conclusions

ChAd63-MVA is a safe and highly immunogenic delivery platform for both alleles of the AMA1 antigen in humans which warrants further efficacy testing. ChAd63-MVA is a promising heterologous prime-boost vaccine strategy that could be applied to numerous other diseases where strong cellular and humoral immune responses are required for protection.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01095055","term_id":"NCT01095055"}}NCT01095055     

Genetic Basis for Rhizobium etli CE3 O-Antigen O-Methylated Residues That Vary According to Growth Conditions

The Rhizobium etli CE3 O antigen is a fixed-length heteropolymer with O methylation being the predominant type of sugar modification. There are two O-methylated residues that occur, on average, once per complete O antigen: a multiply O-methylated terminal fucose and 2-O methylation of a fucose residue within a repeating unit. The amount of the methylated terminal fucose decreases and the amount of 2-O-methylfucose increases when bacteria are grown in the presence of the host plant, Phaseolus vulgaris, or its seed exudates. Insertion mutagenesis was used to identify open reading frames required for the presence of these O-methylated residues. The presence of the methylated terminal fucose required genes wreA, wreB, wreC, wreD, and wreF, whereas 2-O methylation of internal fucoses required the methyltransferase domain of bifunctional gene wreM. Mutants lacking only the methylated terminal fucose, lacking only 2-O methylation, or lacking both the methylated terminal fucose and 2-O methylation exhibited no other lipopolysaccharide structural defects. Thus, neither of these decorations is required for normal O-antigen length, transport, or assembly into the final lipopolysaccharide. This is in contrast to certain enteric bacteria in which the absence of a terminal decoration severely affects O-antigen length and transport. R. etli mutants lacking only the methylated terminal fucose were not altered in symbiosis with host Phaseolus vulgaris, whereas mutants lacking only 2-O-methylfucose exhibited a delay in nodule development during symbiosis. These results support previous conclusions that the methylated terminal fucose is dispensable for symbiosis, whereas 2-O methylation of internal fucoses somehow facilitates early events in symbiosis.O antigens typically constitute the distal portions of lipopolysaccharides (LPS) and help determine the diverse surface characteristics of Gram-negative bacteria. These repeat unit carbohydrate polymers vary tremendously in structure and, as a family, they exhibit all known sugars and sugar modifications, linked in myriad ways forming homopolymers and heteropolymers. Control of polymer length also varies, allowing highly uniform to completely random lengths. Great diversity of O-antigen structures even within a species is well known. Moreover, O antigens of a single strain can vary according to growth and environmental conditions. One such condition is the presence of a multicellular host (5, 18, 36, 40, 42, 44).Rhizobium etli CE3 fixes nitrogen inside root nodules it incites on the common bean Phaseolus vulgaris. The O antigen of its LPS (Fig. ​(Fig.1)1) is essential for bacterial infection during development of this symbiosis (41). In addition, at least two alterations occur in the O antigen when R. etli CE3 is grown in the presence of either the host plant or plant exudates. The content of the multiply O-methylated terminal fucose is decreased (19, 44), whereas the 2-O methylation of internal fucoses (2OMeFuc) increases twofold (Fig. ​(Fig.1)1) (15, 44). In addition to the multiply O-methylated terminal fucose and 2OMeFuc, methylation occurs always on 6-deoxytalose and likely on glucuronic acid to yield 3-O-methyl-6-deoxytalose (3OMe6dTal) and methyl-esterified glucuronyl (MeGlcA) residues (Fig. ​(Fig.1)1) (22); however, the incidence of these methylations is not known to vary with growth condition. The genetics responsible for the variable O methylations and the additions of the residues they modify have not been elucidated.Open in a separate windowFIG. 1.R. etli CE3 O-antigen structure (22). The portion of the LPS conceptually defined as O antigen begins with N-acetyl-quinovosamine (QuiNAc) at the reducing end followed by a mannose (Man) residue and a fucose (Fuc) residue. Attached to this fucose is the repeating unit consisting of one fucose residue, one 3-O-methyl-6-deoxytalose residue (3OMe6dTal), and one glucuronyl methyl ester residue (MeGlcA). The sugars of the repeating unit are added sequentially exactly five times (in most molecules). An O-acetyl group is present in each of the repeating units, but its location is unknown at this time. Growth in TY culture results in one 2-O-methylfucose (2OMeFuc) per O antigen on average (22). The O-antigen backbone is capped with a 2,3-di-O-methylfucose (referred to as the terminal residue in this report) on which additional O methylation at the 4-position is variable as indicated by parentheses. Growth of the bacteria in the presence of the host plant or plant exudates induces the increase of 2-O methylation of internal fucose (2OMeFuc) residues and decreased relative amount of the terminal residue (44).Most mutations affecting the known R. etli CE3 O-antigen structure map to a 28-kb genetic cluster on the chromosome (Fig. ​(Fig.2)2) (previously referred to as lps region α [8, 19, 37, 40, 45]). Genes and mutations within this cluster previously have been given the designations lps (9) and lpe (19). Recently, the new designation wre has been sanctioned by the Bacterial Polysaccharide Gene Database for this genetic cluster and other genes specifically devoted to the R. etli CE3 O antigen, in keeping with the system of nomenclature for bacterial polysaccharide genes (47).Open in a separate windowFIG. 2.R. etli CE3 O-antigen genetic cluster. (A) The R. etli CE3 chromosomal O antigen genetic cluster spans nucleotides 784527 to 812262 of the genome sequence (28) and consists of 25 putative ORFs. ORFs relevant to the present study are enlarged, and the relative locations of mutations are indicated. White triangles indicate mutations created by insertion of antibiotic cassettes, and black triangles indicate mutations created by Tn5 mutagenesis. The strain numbers carrying these mutations are indicated above the triangles. (B) The solid bars represent the extents of R. etli CE3 DNA cloned for complementation analysis. The scale and positions match those of the lower map in panel A.Duelli et al. (19) identified a 3-kb genetic locus that is required for the presence of the 2,3-di-O-methylfucose or 2,3,4-tri-O-methylfucose at the terminus of the O antigen. Now known to be near one end of the O-antigen genetic cluster (Fig. ​(Fig.2),2), the DNA sequence reported by Duelli et al. encompasses nucleotides 807701 to 810147 of the subsequently determined genome sequence (28). Sequence and annotation of the 3-kb locus have since been revised. In place of the four open reading frames (ORFs) suggested previously (19), the current annotation predicts two ORFs: wreA and wreC (Fig. ​(Fig.2).2). The wreA ORF is predicted to encode a methyltransferase (19), but the predicted WreC polypeptide sequence matches no known methyltransferase or glycosyltransferase or any other polypeptide sequence in the database (Fig. ​(Fig.3).3). When it became clear that this locus was part of the larger O-antigen genetic cluster, the nucleotide sequence suggested that three genes contiguous to wreA also might encode functions needed for synthesis and addition of the terminal fucose. The results to be shown bore out predictions of this hypothesis.Open in a separate windowFIG. 3.Conserved domain predictions. Spanning nucleotides 804817 to 810147 of the genome sequence (28), ORFs RHE_CH00766, RHE_CH00767, RHE_CH00768, RHE_CH00769, and RHE_CH00770 were named wreB, wreD, wreF, wreA, and wreC, respectively. Previously, wreF, wreA, and wreC were referred to as nlpe2, lpeA, and nlpe1, respectively (19). ORF RHE_CH00755, spanning nucleotides 791286 to 794093, was named wreM. Predicted positions of conserved domains are indicated by amino acid positions. Abbreviations: GT, conserved glycosyltransferase domain; MT, conserved methyltransferase domain. Gray boxes indicate the predicted transmembrane domains.The gene responsible for the other conditionally variable O-antigen methylation, the 2-O methylation of internal fucose residues (2OMeFuc), had not been identified in prior published work. However, among mutants isolated by random Tn5 mutagenesis, a few had been shown to lack 2OMeFuc entirely (44). We show here that the transposon insertions were located in the bifunctional gene wreM. Furthermore, results of directed insertion mutagenesis confirm two separate enzymatic domains encoded by this gene, with the α domain being required for the 2-O methylation activity and mutation of the other domain resulting in a truncated O antigen. Mutants from the directed mutagenesis that appeared to have no LPS defects other than the lack of 2OMeFuc served as tools to assess the importance of just this structural feature in the symbiosis with P. vulgaris.     

Structural studies on a polysaccharide from Shigella dysenteriae type 7

The polysaccharide obtained from the O-somatic antigen of Shigella dysenteriae type 7 (strain NCTC 519/66) contains d-glucose, d-galactose, and 2-acetamido-2-deoxy-d-glucose in the mole ratios of 2:1:1. From the results of methylation, periodate oxidation, graded hydrolysis, and deamination studies, the structure assigned to the repeating unit of the polysaccharide is as follows.

Oxidation studies with chromium trioxide revealed the nature of the anomeric linkages of some of the sugar residues in the polysaccharide.